Showing papers by "Walter and Eliza Hall Institute of Medical Research published in 2009"
TL;DR: The data suggest that host genetics may be useful for the prediction of drug response, and they also support the investigation of the role of IL28B in the treatment of HCV and in other diseases treated with IFN-α.
Abstract: Hepatitis C virus (HCV) infects 3% of the world's population. Treatment of chronic HCV consists of a combination of PEGylated interferon-alpha (PEG-IFN-alpha) and ribavirin (RBV). To identify genetic variants associated with HCV treatment response, we conducted a genome-wide association study of sustained virological response (SVR) to PEG-IFN-alpha/RBV combination therapy in 293 Australian individuals with genotype 1 chronic hepatitis C, with validation in an independent replication cohort consisting of 555 individuals. We report an association to SVR within the gene region encoding interleukin 28B (IL28B, also called IFNlambda3; rs8099917 combined P = 9.25 x 10(-9), OR = 1.98, 95% CI = 1.57-2.52). IL28B contributes to viral resistance and is known to be upregulated by interferons and by RNA virus infection. These data suggest that host genetics may be useful for the prediction of drug response, and they also support the investigation of the role of IL28B in the treatment of HCV and in other diseases treated with IFN-alpha.
1,858 citations
TL;DR: ELDA is a software application for limiting dilution analysis (LDA), with particular attention to the needs of stem cell assays, which is the first limiting dilutions analysis software to provide meaningful confidence intervals for all LDA data sets, including those with 0% or 100% responses.
1,645 citations
TL;DR: It is found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro, and the findings suggest that an aberrant luminalprogenitor population is a target for transformation in BRCa1-associated basal tumors.
Abstract: Basal-like breast cancers arising in women carrying mutations in the BRCA1 gene, encoding the tumor suppressor protein BRCA1, are thought to develop from the mammary stem cell. To explore early cellular changes that occur in BRCA1 mutation carriers, we have prospectively isolated distinct epithelial subpopulations from normal mammary tissue and preneoplastic specimens from individuals heterozygous for a BRCA1 mutation. We describe three epithelial subsets including basal stem/progenitor, luminal progenitor and mature luminal cells. Unexpectedly, we found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro. Moreover, gene expression profiling revealed that breast tissue heterozygous for a BRCA1 mutation and basal breast tumors were more similar to normal luminal progenitor cells than any other subset, including the stem cell-enriched population. The c-KIT tyrosine kinase receptor (encoded by KIT) emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA1-associated preneoplastic tissue and tumors. Our findings suggest that an aberrant luminal progenitor population is a target for transformation in BRCA1-associated basal tumors .
1,339 citations
TL;DR: The exacerbated release of exosomes in tumor cells, as evidenced by their increased levels in blood during the late stage of a disease and their overexpression of certain tumor cell biomarkers, suggests an important role of exOSomes in diagnosis and biomarker studies.
Abstract: Exosomes are 40-100-nm diameter membrane vesicles of endocytic origin that are released by most cell types upon fusion of multivesicular bodies with the plasma membrane, presumably as a vehicle for cell-free intercellular communication. While early studies focused on their secretion from diverse cell types in vitro, exosomes have now been identified in body fluids such as urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, saliva and blood. Exosomes have pleiotropic biological functions, including immune response, antigen presentation, intracellular communication and the transfer of RNA and proteins. While they have also been implicated in the transport and propagation of infectious cargo, such as prions, and retroviruses, including HIV, suggesting a role in pathological situations, recent studies suggest that the presence of such infectious cargo may be artefacts of exosome-purification strategies. Improvements in mass spectrometry-based proteomic tools, both hardware and software, coupled with improved purification schemes for exosomes, has allowed more in-depth proteome analyses, contributing immensely to our understanding of the molecular composition of exosomes. Proteomic cataloguing of exosomes from diverse cell types has revealed a common set of membrane and cytosolic proteins, suggesting the evolutionary importance of these membrane particles. Additionally, exosomes express an array of proteins that reflect the originating host cell. Recent findings that exosomes contain inactive forms of both mRNA and microRNA that can be transferred to another cell and be functional in that new environment, have initiated many microRNA profiling studies of exosomes circulating in blood. These studies highlight the potential of exosomal microRNA profiles for use as diagnostic biomarkers of disease through a noninvasive blood test. The exacerbated release of exosomes in tumor cells, as evidenced by their increased levels in blood during the late stage of a disease and their overexpression of certain tumor cell biomarkers, suggests an important role of exosomes in diagnosis and biomarker studies. The aim of this article is to provide a brief overview of exosomes, including methods used to isolate and characterize exosomes. New advances in proteomic methods, and both mass spectrometry hardware and informatics tools will be covered briefly.
960 citations
TL;DR: The recognition that cell death can occur by genetically controlled processes has enabled advances in unraveling the mechanisms of many diseases and facilitated development of pharmacologic agents that initiate or inhibit programmed cell death.
Abstract: One of the abiding mysteries of all multi-cellular organisms is the requirement for controlled death —apoptosis — of unwanted cells. It has been estimated that without apoptosis an 80 year old person would have two tons of bone marrow and lymph nodes and an intestine 16 kilometers long.1 Progress in defining pathways of apoptosis has revealed complex interconnections between various cell death programs that may affect the treatment of a wide range of diseases.2–10 This article reviews advances in our understanding of mechanisms of cell death and highlights current and potential therapies based upon these concepts.
Perhaps the most widely used classification of mammalian cell death consists of two types: apoptosis and necrosis.3,4,11 Autophagy, which has recently been proposed as a third distinct mode of cell death, is a process by which cells generate energy and metabolites by digesting organelles or macromolecules.12–15. Normally, autophagy allows a starving cell, or a cell deprived of growth factors to survive.12–15 Ultimately, however, cells deprived of nutrients for extended periods will digest all available substrates and die an ‘autophagy-associated cell death’. Distinctions between apoptosis, necrosis, and autophagy entail differences in mode-specific or selective morphologic, biochemical, and molecular attributes (Fig. 1).3,4,11
Figure 1
Schematic diagram showing 3 possible pathways of cell death
An important concept embodied in part by these attributes is “programmed” cell death. Cell death is “programmed” if it is genetically controlled. The two fundamental types of programmed cell death are apoptosis and autophagy-associated cell death.3,12 The recognition that cell death can occur by genetically controlled processes has enabled advances in unraveling the mechanisms of many diseases. As a result, we now have improved knowledge of the initiation of cell death programs and the relevant signaling pathways. This information has facilitated development of pharmacologic agents that initiate or inhibit programmed cell death.6–8,16 Moreover, there is now evidence that necrosis, traditionally considered an accidental form of cell death, can, in certain instances, be initiated or modulated under programmed control mechanisms.17–21
939 citations
TL;DR: Current understanding of Fas-induced apoptosis signaling is described and experimental strategies for future advances are proposed.
847 citations
TL;DR: The results indicate that Treg cells use components of the transcriptional machinery, promoting a particular type of effector CD4+ T cell differentiation, to efficiently restrain the corresponding type of the immune response.
Abstract: In the course of infection or autoimmunity, particular transcription factors orchestrate the differentiation of T(H)1, T(H)2 or T(H)17 effector cells, the responses of which are limited by a distinct lineage of suppressive regulatory T cells (T(reg)). T(reg) cell differentiation and function are guided by the transcription factor Foxp3, and their deficiency due to mutations in Foxp3 results in aggressive fatal autoimmune disease associated with sharply augmented T(H)1 and T(H)2 cytokine production. Recent studies suggested that Foxp3 regulates the bulk of the Foxp3-dependent transcriptional program indirectly through a set of transcriptional regulators serving as direct Foxp3 targets. Here we show that in mouse T(reg) cells, high amounts of interferon regulatory factor-4 (IRF4), a transcription factor essential for T(H)2 effector cell differentiation, is dependent on Foxp3 expression. We proposed that IRF4 expression endows T(reg) cells with the ability to suppress T(H)2 responses. Indeed, ablation of a conditional Irf4 allele in T(reg) cells resulted in selective dysregulation of T(H)2 responses, IL4-dependent immunoglobulin isotype production, and tissue lesions with pronounced plasma cell infiltration, in contrast to the mononuclear-cell-dominated pathology typical of mice lacking T(reg) cells. Our results indicate that T(reg) cells use components of the transcriptional machinery, promoting a particular type of effector CD4(+) T cell differentiation, to efficiently restrain the corresponding type of the immune response.
835 citations
TL;DR: A compendium for proteins and RNA molecules identified in exosomes, first of its kind and freely available to the scientific community through the web, is described and believed that this community resource will be of great biological importance for any future exosome analyses.
Abstract: Exosomes, membrane microvesicles (40-100 nm) secreted by most cell types, can be isolated in several ways while characterizing them is heavily based on electron microscopy and, most importantly, the identification of exosome marker proteins. Researchers rely on the identification of certain exosomal marker proteins including Alix, CD9 and CD63 to confirm the presence of exosomes in their preparations. An evolutionary-conserved set of protein molecules have been identified in most exosomes studied to date. However, with the complexity of tissue/cell type-specific proteins being incorporated in the exosomes, some of these so-called exosomal markers are not always present in all the exosomes. The presence of tissue/cell type-specific proteins in exosomes allows researchers to isolate them using immunoaffinity capture methods. A compendium for exosomal proteomes will aid researchers in identifying proteins that were more commonly found in various exosomes (exosome markers) and those that are specific to certain tissue/cell type-derived exosomes. Here, we describe ExoCarta, a compendium for proteins and RNA molecules identified in exosomes. ExoCarta is first of its kind and the resource is freely available to the scientific community through the web (http://exocarta.ludwig.edu.au). We believe that this community resource will be of great biological importance for any future exosome analyses.
793 citations
TL;DR: It is shown that CD103+ DCs were the migratory subset most efficient at processing viral antigens into the major histocompatibility complex class I pathway, potentially through cross-presentation.
Abstract: Skin-derived dendritic cells (DCs) include Langerhans cells, classical dermal DCs and a langerin-positive CD103(+) dermal subset. We examined their involvement in the presentation of skin-associated viral and self antigens. Only the CD103(+) subset efficiently presented antigens of herpes simplex virus type 1 to naive CD8(+) T cells, although all subsets presented these antigens to CD4(+) T cells. This showed that CD103(+) DCs were the migratory subset most efficient at processing viral antigens into the major histocompatibility complex class I pathway, potentially through cross-presentation. This was supported by data showing only CD103(+) DCs efficiently cross-presented skin-derived self antigens. This indicates CD103(+) DCs are the main migratory subtype able to cross-present viral and self antigens, which identifies another level of specialization for skin DCs.
685 citations
TL;DR: A number of overarching structural, functional, and evolutionary generalities of the protein families from which these toxins have been frequently recruited are discussed and a revised and expanded working definition for venom is proposed.
Abstract: Throughout evolution, numerous proteins have been convergently recruited into the venoms of various animals, including centipedes, cephalopods, cone snails, fish, insects (several independent venom systems), platypus, scorpions, shrews, spiders, toxicoferan reptiles (lizards and snakes), and sea anemones. The protein scaffolds utilized convergently have included AVIT/colipase/prokineticin, CAP, chitinase, cystatin, defensins, hyaluronidase, Kunitz, lectin, lipocalin, natriuretic peptide, peptidase S1, phospholipase A2, sphingomyelinase D, and SPRY. Many of these same venom protein types have also been convergently recruited for use in the hematophagous gland secretions of invertebrates (e.g., fleas, leeches, kissing bugs, mosquitoes, and ticks) and vertebrates (e.g., vampire bats). Here, we discuss a number of overarching structural, functional, and evolutionary generalities of the protein families from which these toxins have been frequently recruited and propose a revised and expanded working definition...
654 citations
TL;DR: This work presents a method, t-tests relative to a threshold (TREAT), that allows researchers to test formally the hypothesis (with associated p-values) that the differential expression in a microarray experiment is greater than a given threshold.
Abstract: Motivation: Statistical methods are used to test for the differential expression of genes in microarray experiments. The most widely used methods successfully test whether the true differential expression is different from zero, but give no assurance that the differences found are large enough to be biologically meaningful.
Results: We present a method, t-tests relative to a threshold (TREAT), that allows researchers to test formally the hypothesis (with associated p-values) that the differential expression in a microarray experiment is greater than a given (biologically meaningful) threshold. We have evaluated the method using simulated data, a dataset from a quality control experiment for microarrays and data from a biological experiment investigating histone deacetylase inhibitors. When the magnitude of differential expression is taken into account, TREAT improves upon the false discovery rate of existing methods and identifies more biologically relevant genes.
Availability: R code implementing our methods is contributed to the software package limma available at http://www.bioconductor.org.
Contact: smyth@wehi.edu.au
TL;DR: Transcript length bias for calling differentially expressed genes is a general feature of current protocols for RNA-seq technology and has implications for the ranking of differentially expression genes, and in particular may introduce bias in gene set testing for pathway analysis and other multi-gene systems biology analyses.
Abstract: Several recent studies have demonstrated the effectiveness of deep sequencing for transcriptome analysis (RNA-seq) in mammals. As RNA-seq becomes more affordable, whole genome transcriptional profiling is likely to become the platform of choice for species with good genomic sequences. As yet, a rigorous analysis methodology has not been developed and we are still in the stages of exploring the features of the data. We investigated the effect of transcript length bias in RNA-seq data using three different published data sets. For standard analyses using aggregated tag counts for each gene, the ability to call differentially expressed genes between samples is strongly associated with the length of the transcript. Transcript length bias for calling differentially expressed genes is a general feature of current protocols for RNA-seq technology. This has implications for the ranking of differentially expressed genes, and in particular may introduce bias in gene set testing for pathway analysis and other multi-gene systems biology analyses. This article was reviewed by Rohan Williams (nominated by Gavin Huttley), Nicole Cloonan (nominated by Mark Ragan) and James Bullard (nominated by Sandrine Dudoit).
Walter and Eliza Hall Institute of Medical Research1, Millennium Institute2, Griffith University3, University of Oxford4, University of Queensland5, University of Tasmania6, Royal Melbourne Hospital7, University of Melbourne8, John Hunter Hospital9, University of Newcastle10, Nepean Hospital11, University of Otago12, University of Auckland13, Box Hill Hospital14, Sir Charles Gairdner Hospital15, Geelong Hospital16, Royal Brisbane and Women's Hospital17, Flinders University18, University College Dublin19
TL;DR: To identify multiple sclerosis (MS) susceptibility loci, a genome-wide association study in 1,618 cases and used shared data for 3,413 controls and observed a statistical interaction between SNPs in EVI5-RPL5 and HLA-DR15.
Abstract: To identify multiple sclerosis (MS) susceptibility loci, we conducted a genome-wide association study (GWAS) in 1,618 cases and used shared data for 3,413 controls. We performed replication in an independent set of 2,256 cases and 2,310 controls, for a total of 3,874 cases and 5,723 controls. We identified risk-associated SNPs on chromosome 12q13-14 (rs703842, P = 5.4 x 10(-11); rs10876994, P = 2.7 x 10(-10); rs12368653, P = 1.0 x 10(-7)) and upstream of CD40 on chromosome 20q13 (rs6074022, P = 1.3 x 10(-7); rs1569723, P = 2.9 x 10(-7)). Both loci are also associated with other autoimmune diseases. We also replicated several known MS associations (HLA-DR15, P = 7.0 x 10(-184); CD58, P = 9.6 x 10(-8); EVI5-RPL5, P = 2.5 x 10(-6); IL2RA, P = 7.4 x 10(-6); CLEC16A, P = 1.1 x 10(-4); IL7R, P = 1.3 x 10(-3); TYK2, P = 3.5 x 10(-3)) and observed a statistical interaction between SNPs in EVI5-RPL5 and HLA-DR15 (P = 0.001).
TL;DR: It is shown that the transcription factor Blimp-1, a crucial regulator of plasma cell differentiation, was required for CD8(+) T cells to differentiate into functional killer T cells in response to influenza virus.
TL;DR: The results show that XIAP is the critical discriminator between type I and type II apoptosis signalling and suggest that IAP inhibitors should be used with caution in cancer patients with underlying liver conditions.
Abstract: FAS (also called APO-1 and CD95) and its physiological ligand, FASL, regulate apoptosis of unwanted or dangerous cells, functioning as a guardian against autoimmunity and cancer development. Distinct cell types differ in the mechanisms by which the 'death receptor' FAS triggers their apoptosis. In type I cells, such as lymphocytes, activation of 'effector caspases' by FAS-induced activation of caspase-8 suffices for cell killing, whereas in type II cells, including hepatocytes and pancreatic beta-cells, caspase cascade amplification through caspase-8-mediated activation of the pro-apoptotic BCL-2 family member BID (BH3 interacting domain death agonist) is essential. Here we show that loss of XIAP (X-chromosome linked inhibitor of apoptosis protein) function by gene targeting or treatment with a second mitochondria-derived activator of caspases (SMAC, also called DIABLO; direct IAP-binding protein with low pI) mimetic drug in mice rendered hepatocytes and beta-cells independent of BID for FAS-induced apoptosis. These results show that XIAP is the critical discriminator between type I and type II apoptosis signalling and suggest that IAP inhibitors should be used with caution in cancer patients with underlying liver conditions.
TL;DR: This work has identified in Plasmodium falciparum a translocon of exported proteins (PTEX), which is located in the vacuole membrane and offers a new avenue for therapeutic intervention.
Abstract: Several hundred malaria parasite proteins are exported beyond an encasing vacuole and into the cytosol of the host erythrocyte, a process that is central to the virulence and viability of the causative Plasmodium species. The trafficking machinery responsible for this export is unknown. Here we identify in Plasmodium falciparum a translocon of exported proteins (PTEX), which is located in the vacuole membrane. The PTEX complex is ATP-powered, and comprises heat shock protein 101 (HSP101; a ClpA/B-like ATPase from the AAA+ superfamily, of a type commonly associated with protein translocons), a novel protein termed PTEX150 and a known parasite protein, exported protein 2 (EXP2). EXP2 is the potential channel, as it is the membrane-associated component of the core PTEX complex. Two other proteins, a new protein PTEX88 and thioredoxin 2 (TRX2), were also identified as PTEX components. As a common portal for numerous crucial processes, this translocon offers a new avenue for therapeutic intervention.
TL;DR: Developments in molecular transfection technology, including the ability to generate deletion mutants and to introduce fluorescent reporter proteins that track the locations and dynamics of parasite proteins, have increased understanding of the processes and machinery for export of proteins in P. falciparum-infected erythrocytes and has provided insights into the functions of the parasite protein exportome.
Abstract: Exported proteins of the malaria parasite Plasmodium falciparum interact with proteins of the erythrocyte membrane and induce substantial changes in the morphology, physiology and function of the host cell. These changes underlie the pathology that is responsible for the deaths of 1-2 million children every year due to malaria infections. The advent of molecular transfection technology, including the ability to generate deletion mutants and to introduce fluorescent reporter proteins that track the locations and dynamics of parasite proteins, has increased our understanding of the processes and machinery for export of proteins in P. falciparum-infected erythrocytes and has provided us with insights into the functions of the parasite protein exportome. We review these developments, focusing on parasite proteins that interact with the erythrocyte membrane skeleton or that promote delivery of the major virulence protein, PfEMP1, to the erythrocyte membrane.
TL;DR: It is shown in a rodent malaria model that FAS II enzymes localize to the sporozoite and liver stage apicoplast and deletion of FabI in the human malaria parasite Plasmodium falciparum did not show a reduction in asexual blood stage replication in vitro, indicating that malaria parasites depend on the intrinsic FASII pathway only at one specific life cycle transition point, from liver to blood.
Abstract: Intracellular malaria parasites require lipids for growth and replication. They possess a prokaryotic type II fatty acid synthesis (FAS II) pathway that localizes to the apicoplast plastid organelle and is assumed to be necessary for pathogenic blood stage replication. However, the importance of FAS II throughout the complex parasite life cycle remains unknown. We show in a rodent malaria model that FAS II enzymes localize to the sporozoite and liver stage apicoplast. Targeted deletion of FabB/F, a critical enzyme in fatty acid synthesis, did not affect parasite blood stage replication, mosquito stage development and initial infection in the liver. This was confirmed by knockout of FabZ, another critical FAS II enzyme. However, FAS II-deficient Plasmodium yoelii liver stages failed to form exo-erythrocytic merozoites, the invasive stage that first initiates blood stage infection. Furthermore, deletion of FabI in the human malaria parasite Plasmodium falciparum did not show a reduction in asexual blood stage replication in vitro. Malaria parasites therefore depend on the intrinsic FAS II pathway only at one specific life cycle transition point, from liver to blood.
TL;DR: It is demonstrated that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFAsL appears to promote autoimunity and tumorigenesis through non-apoptotic activities.
Abstract: Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasL(Deltas/Deltas)) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasL(Deltam/Deltam)) could not kill cells through Fas activation. FasL(Deltam/Deltam) mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasL(gld/gld) mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasL(Deltam/Deltam) mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasL(gld/gld) mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.
TL;DR: Genome-wide ChIP-Seq mapping of STAT3- and IRF4-binding sites showed that most regions with IL-21-induced STAT3 binding also bound IRf4 in vivo and furthermore revealed that the noncanonical TTCnnnTAA GAS motif critical in Prdm1 was broadly used forSTAT3 binding.
McGill University1, Institute for Systems Biology2, University of British Columbia3, National Institutes of Health4, Invitrogen5, Allergan6, Northeastern University7, Ruhr University Bochum8, Massachusetts Institute of Technology9, Discovery Institute10, Fred Hutchinson Cancer Research Center11, Georgetown University12, University of Gothenburg13, Harvard University14, Thermo Fisher Scientific15, Laval University16, Walter and Eliza Hall Institute of Medical Research17, University of Toronto18, Scripps Research Institute19, University of Alberta20, University of California, Los Angeles21, University College Dublin22, University of Michigan23, University of Pittsburgh24, University of Victoria25, University of Western Ontario26, Wistar Institute27, Yamaguchi University28, Yonsei University29, Agilent Technologies30, Applied Biosystems31, Waters Corporation32
TL;DR: Central analysis determined missed identifications, environmental contamination, database matching and curation of protein identifications as sources of problems in liquid chromatography–mass spectrometry–based proteomics.
Abstract: We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography-mass spectrometry-based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry-based proteomics.
TL;DR: The reported key step leading to Bak homo-oligomerisation following an apoptotic stimulus involves transient exposure of the Bak BH3 domain before it binds to the hydrophobic groove of another activated Bak molecule to form a novel symmetric dimer.
Abstract: Mitochondrial outer membrane permeabilisation (MOMP) is the point of no return in many forms of apoptotic cell death. The killing effect of MOMP is twofold; it both initiates a proteolytic cascade of pro-apoptotic enzymes and damages mitochondrial function. Accordingly, prevention of MOMP can rescue cells from death. It is clear that either Bak or Bax, which are Bcl-2 family members, are required for MOMP to occur; however, the pore complexes that are formed by Bak and Bax remain poorly defined in terms of their composition, size, number and structure, as well as the mechanism by which they are regulated by other Bcl-2 family members. We recently reported that a key step leading to Bak homo-oligomerisation following an apoptotic stimulus involves transient exposure of the Bak BH3 domain before it binds to the hydrophobic groove of another activated Bak molecule to form a novel symmetric dimer. To form the higher-order oligomers that probably constitute the apoptotic pore complex, Bak dimers then interact via regions away from the BH3 domain and groove. The BH3:groove interaction within Bak homodimers supports a general model to explain the associations between Bcl-2 family members. In this Commentary, we discuss the implications of these findings for the regulation of apoptosis by Bcl-2 family proteins.
TL;DR: 2 distinct pathways regulating the procoagulant function of platelets are shown, one of which is Bak/Bax- and caspase-dependent and the other is agonist-induced, which was completely eliminated by extracellular calcium chelators or inhibitors of platelet activation.
TL;DR: Characterisation of the P. falciparum silent information regulator's A and B and their involvement in mutual exclusion and silencing of the var gene repertoire highlights the pivotal but distinct role for both PfSir2 paralogues in epigenetic silencing and the control of pathogenicity of malaria infection.
Abstract: Cytoadherance of Plasmodium falciparum-infected erythrocytes in the brain, organs and peripheral microvasculature is linked to morbidity and mortality associated with severe malaria. Parasite-derived P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) molecules displayed on the erythrocyte surface are responsible for cytoadherance and undergo antigenic variation in the course of an infection. Antigenic variation of PfEMP1 is achieved by in situ switching and mutually exclusive transcription of the var gene family, a process that is controlled by epigenetic mechanisms. Here we report characterisation of the P. falciparum silent information regulator's A and B (PfSir2A and PfSir2B) and their involvement in mutual exclusion and silencing of the var gene repertoire. Analysis of P. falciparum parasites lacking either PfSir2A or PfSir2B shows that these NAD(+)-dependent histone deacetylases are required for silencing of different var gene subsets classified by their conserved promoter type. We also demonstrate that in the absence of either of these molecules mutually exclusive expression of var genes breaks down. We show that var gene silencing originates within the promoter and PfSir2 paralogues are involved in cis spreading of silenced chromatin into adjacent regions. Furthermore, parasites lacking PfSir2A but not PfSir2B have considerably longer telomeric repeats, demonstrating a role for this molecule in telomeric end protection. This work highlights the pivotal but distinct role for both PfSir2 paralogues in epigenetic silencing of P. falciparum virulence genes and the control of pathogenicity of malaria infection.
TL;DR: Results indicate a protective function for platelets in the early stages of erythrocytic infection distinct from their role in cerebral malaria.
Abstract: Platelets play a critical role in the pathogenesis of malarial infections by encouraging the sequestration of infected red blood cells within the cerebral vasculature. But platelets also have well-established roles in innate protection against microbial infections. We found that purified human platelets killed Plasmodium falciparum parasites cultured in red blood cells. Inhibition of platelet function by aspirin and other platelet inhibitors abrogated the lethal effect human platelets exert on P. falciparum parasites. Likewise, platelet-deficient and aspirin-treated mice were more susceptible to death during erythrocytic infection with Plasmodium chabaudi. Both mouse and human platelets bind malarial-infected red cells and kill the parasite within. These results indicate a protective function for platelets in the early stages of erythrocytic infection distinct from their role in cerebral malaria.
TL;DR: It is shown that P. falciparum heterochromatin protein 1 (PfHP1) binds specifically to H3K9me3 but not to other repressive histone methyl marks, which identifies PfHP1 as a major effector of virulence gene silencing and phenotypic variation.
Abstract: Epigenetic processes are the main conductors of phenotypic variation in eukaryotes. The malaria parasite Plasmodium falciparum employs antigenic variation of the major surface antigen PfEMP1, encoded by 60 var genes, to evade acquired immune responses. Antigenic variation of PfEMP1 occurs through in situ switches in mono-allelic var gene transcription, which is PfSIR2-dependent and associated with the presence of repressive H3K9me3 marks at silenced loci. Here, we show that P. falciparum heterochromatin protein 1 (PfHP1) binds specifically to H3K9me3 but not to other repressive histone methyl marks. Based on nuclear fractionation and detailed immuno-localization assays, PfHP1 constitutes a major component of heterochromatin in perinuclear chromosome end clusters. High-resolution genome-wide chromatin immuno-precipitation demonstrates the striking association of PfHP1 with virulence gene arrays in subtelomeric and chromosome-internal islands and a high correlation with previously mapped H3K9me3 marks. These include not only var genes, but also the majority of P. falciparum lineage-specific gene families coding for exported proteins involved in host-parasite interactions. In addition, we identified a number of PfHP1-bound genes that were not enriched in H3K9me3, many of which code for proteins expressed during invasion or at different life cycle stages. Interestingly, PfHP1 is absent from centromeric regions, implying important differences in centromere biology between P. falciparum and its human host. Over-expression of PfHP1 results in an enhancement of variegated expression and highlights the presence of well-defined heterochromatic boundaries. In summary, we identify PfHP1 as a major effector of virulence gene silencing and phenotypic variation. Our results are instrumental for our understanding of this widely used survival strategy in unicellular pathogens.
TL;DR: Substantial evidence indicates that antibodies to Plasmodium falciparum merozoite antigens play a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear and the factors determining subclass responses in vivo are poorly understood.
Abstract: Substantial evidence indicates that antibodies to Plasmodium falciparum merozoite antigens play a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear. Different malaria antigens induce distinct immunoglobulin G (IgG) subclass responses, but the importance of different responses in protective immunity from malaria is not known and the factors determining subclass responses in vivo are poorly understood. We examined IgG and IgG subclass responses to the merozoite antigens MSP1-19 (the 19-kDa C-terminal region of merozoite surface protein 1), MSP2 (merozoite surface protein 2), and AMA-1 (apical membrane antigen 1), including different polymorphic variants of these antigens, in a longitudinal cohort of children in Papua New Guinea. IgG1 and IgG3 were the predominant subclasses of antibodies to each antigen, and all antibody responses increased in association with age and exposure without evidence of increasing polarization toward one subclass. The profiles of IgG subclasses differed somewhat for different alleles of MSP2 but not for different variants of AMA-1. Individuals did not appear to have a propensity to make a specific subclass response irrespective of the antigen. Instead, data suggest that subclass responses to each antigen are generated independently among individuals and that antigen properties, rather than host factors, are the major determinants of IgG subclass responses. High levels of AMA-1-specific IgG3 and MSP1-19-specific IgG1 were strongly predictive of a reduced risk of symptomatic malaria and high-density P. falciparum infections. However, no antibody response was significantly associated with protection from parasitization per se. Our findings have major implications for understanding human immunity and for malaria vaccine development and evaluation.
TL;DR: Comparisons between prototherian and therian mammals provide strong support for the host defence hypothesis and show that the platypus has significantly fewer repeats of certain classes in the regions of the genome that have become imprinted in therian mammal.
Abstract: Background: Genomic imprinting is an epigenetic phenomenon that results in monoallelic gene expression. Many hypotheses have been advanced to explain why genomic imprinting evolved in mammals, but few have examined how it arose. The host defence hypothesis suggests that imprinting evolved from existing mechanisms within the cell that act to silence foreign DNA elements that insert into the genome. However, the changes to the mammalian genome that accompanied the evolution of imprinting have been hard to define due to the absence of large scale genomic resources between all extant classes. The recent release of the platypus genome has provided the first opportunity to perform comparisons between prototherian (monotreme; which appear to lack imprinting) and therian (marsupial and eutherian; which have imprinting) mammals. Results: We compared the distribution of repeat elements known to attract epigenetic silencing across the entire genome from monotremes and therian mammals, particularly focusing on the orthologous imprinted regions. There is a significant accumulation of certain repeat elements within imprinted regions of therian mammals compared to the platypus. Conclusions: Our analyses show that the platypus has significantly fewer repeats of certain classes in the regions of the genome that have become imprinted in therian mammals. The accumulation of repeats, especially long terminal repeats and DNA elements, in therian imprinted genes and gene clusters is coincident with, and may have been a potential driving force in, the development of mammalian genomic imprinting. These data provide strong support for the host defence hypothesis.
TL;DR: Recent developments in the phenotypic characterisation of memory B cells are examined, with an emphasis on the definition and functional properties of memoryB‐cell subsets in humans, and recent advances in the understanding of germinal center B‐cell differentiation are considered.
Abstract: Immunological memory is the phenomenon whereby B and T cells have the unique ability to respond with heightened kinetics and efficacy to subsequent encounter with Ag relative to the initial exposure. In this review, we examine recent developments in the phenotypic characterisation of memory B cells, with an emphasis on the definition and functional properties of memory B-cell subsets in humans. Gene expression differences are also considered in light of the unique functional and survival properties of memory B cells, and mutations that alter memory formation and function are also examined. Finally, we consider recent advances in the understanding of germinal center B-cell differentiation through analysis of transcription factor networks operating in these B cells.
TL;DR: Video microscopy is used to address the kinetics of RBC invasion in the human malaria parasite Plasmodium falciparum and provides a morphological and kinetic framework onto which the invasion-associated physiological and molecular events can be overlaid.